DNA polymerase beta: Pre-steady-state kinetic analyses of dATP alpha S stereoselectivity and alteration of the stereoselectivity by various metal ions and by site-directed mutagenesis

The first pre-steady-state kinetic analysis of the stereoselectivity of a D NA polymerase, Pol beta from rat brain, toward Rp and Sp isomers of dATP al phaS, and alteration of the stereoselectivity by various metal ions and by site-directed mutagenesis are reported. Diastereomers of dATP alphaS were s ynthesized by enzymatic methods to >98% purity. The rate of polymerization (k(pol)) and the apparent dissociation constant (K-d,K-app) were measured w ith dATP, Rp-dATP alphaS, and Sp-dATP alphaS in the presence of Mg2+, Mn2+, or Cd2+. The results indicate that wild type (WT) polymerase (Pol) beta ca n incorporate both Sp- and Rp-dATP alphaS in the presence of Mg2+, but Sp i s the preferred isomer. The stereoselectivity, defined as (k(pol)/ K-d)(Sp) /(k(pol)/K-d)(Rp) (abbreviated Sp/Rp ratio), is 57.5 in the presence of Mg2 +. When Mg2+ was substituted with Mn2+ and Cd2+, the Sp/Rp ratio decreased to 7.6 and 21, respectively. These results are discussed in relation to the crystal structures of various Pol beta complexes, as well as previous stea dy-state kinetic studies of other DNA polymerases. In addition, the D276R m utant was designed to introduce a potential extra hydrogen bonding interact ion between the arginine side chain and the pro-Sp oxygen of the alpha -pho sphate of dNTP. The kinetic data of the D276R mutant showed a pronounced re laxation of stereoselectivity of dATP alphaS (Sp/Rp ratio = 1.5, 3.7, and 1 .5 for Mg2+, Mn2+, and Cd2+, respectively). Furthermore, the D276R mutant s howed a 5-fold enhanced reactivity toward Rp-dATP alphaS relative to WT Pol beta, suggesting that this mutant Pol beta can be used to incorporate Rp-d NTP alphaS into DNA oligomers.